Ras-Mediated Activation of the Raf Family Kinases

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Elizabeth M. Terrell and Deborah K. Morrison

Laboratory of Cell and Developmental Signaling, NCI-Frederick, Frederick, Maryland 21702 Correspondence: [email protected]

The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade com- ponents, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.

embers of the Raf serine/threonine kinase men and Marais 2007), and germline mutations Mfamily are the initiating enzymes in the in either B-Raf or C-Raf can be causative for three-tiered extracellular signal-regulated kinase certain developmental disorders in the Rasopa- (ERK) cascade, and in mammalian cells, there thies spectrum (Tartaglia et al. 2011). are three Raf proteins, A-Raf, B-Raf, and C-Raf Despite their critical function in cell signal- (also known as Raf-1) (Wellbrock et al. 2004). ing, determining how Raf activity is regulated The Raf kinases were ﬁrst discovered when the has been a challenging task—resulting from raf-1 gene was identiﬁed as the cellular counter- the complexity of the Raf activation process. In part of the murine retroviral oncogene, v-raf this article, we will review the molecular mech-

www.perspectivesinmedicine.org (Rapp et al. 1983). Later, this kinase family anisms involved in Raf kinase regulation. Our rose to prominence when they were found to discussion will include an examination of the be direct effectors of activated Ras, mediating presignaling inactive state of the Rafs as well as signal transmission from Ras to the downstream the events that contribute to Raf kinase activa- kinases MEK and ERK (Marshall 1996). Not tion, with a focus on the crucial role that the Ras surprisingly, given their central position in GTPases play in promoting Raf activation under such a major signaling pathway, mutations in normal growth conditions as well as in human the Raf kinases also contribute to human disease disease states. Whenever possible, we will also states. In particular, somatic mutations in B-Raf comment on the regulatory differences that have are important drivers of human cancer (Dho- been identiﬁed for the individual Raf members,

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E.M. Terrell and D.K. Morrison

and we will conclude with a description of the localize to the cytosol as monomers (Nan et al. mechanisms that attenuate Raf activity follow- 2013). Before pathway activation, the Raf mono- ing signal transmission. mers are maintained in a stable, presignaling inactive state through multiple regulatory mechanisms, including autoinhibition, phosphoryla- THE RAF PRESIGNALING INACTIVE STATE tion of negative regulatory sites, and binding of All members of the Raf kinase family contain inhibitory proteins (Figs. 1B and 2). three conserved regions, CR1, CR2, and CR3, and each Raf protein can be divided into two Raf Autoinhibition functional domains—an amino-terminal regulatory domain and a carboxy-terminal catalytic The ﬁrst indication that the Raf amino-terminal domain (Fig. 1A) (Daum et al. 1994). Both the domain might possess autoinhibitory activity CR1, which consists of a Ras-binding domain came from the observation that this domain is (RBD) and a cysteine-rich domain (CRD), and absent in the oncogenic v-Raf protein (Rapp the CR2, a region rich in serine/threonine resi- et al. 1983). Subsequently, it was shown that dues, are found within the amino-terminal reg- deletion of the amino-terminal domain could ulatory domain, whereas the CR3 comprises the convert any of the mammalian Raf proteins carboxy-terminal kinase domain. The Raf pro- into constitutively active kinases capable of in- teins do not contain any intrinsic subcellular ducing cellular transformation (Stanton et al. localization motifs, and in quiescent cells, they 1989; Heidecker et al. 1990). Further studies in

A Regulatory domain Kinase domain

CR1 CR2 CR3

RBD CRD S/T Kinase domain A-Raf

RBD CRD S/T Kinase domain B-Raf

RBD CRD S/T Kinase domain C-Raf

B 14-3-3 14-3-3 www.perspectivesinmedicine.org Ras Hsp90 complex RKIP (CR2) (C )

RBD CRD S/T Kinase domain

A/B/C-Raf and KSRs

Membrane lipids MEK

Figure 1. Raf domain structure and protein interactions. (A) The Raf kinases can be divided into two functional domains: an amino-terminal regulatory domain and a carboxy-terminal kinase domain and they contain three conserved regions (CRs): CR1, which contains a Ras-binding domain (RBD) and a cysteine-rich domain (CRD); CR2, an area rich in serine/threonine residues (S/T); and CR3, which contains the protein kinase domain. (B) Numerous Raf-binding partners have been identiﬁed.

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Ras-Mediated Activation of the Raf Family Kinases

14-3-3 Activating: N-region AS (+) site

RBD CRD S/T Kinase domain

Inhibitory: pSP pS/TP pS/TP 14-3-3 P-loop AS (–) site

CK2, PAK, SFK Raf AMPK, PKA PKC

N-region Activation segment (AS) 14-3-3 C site A–Raf: GYRDSGYYWEV DFGLATVKTRWSGAQPLEQPSGSVLWMAAE IERSASEPSL B–Raf: GRRDSSDDWEI DFGLATVKSRWSGSHQFEQLSGSILWMAPE IHRSASEPSL C–Raf: GQRDSSYYWEI DFGLATVKSRWSGSQQVEQPTGSVLWMAPE INRSASEPSL

PKA, AKT, LATS1 Raf ERK, JNK

14-3-3 CR2-site P-loop pS/TP A–Raf: RIRSTSTPNV RIGTGSFG T181,T253,S257,S259 B–Raf: RDRSSSAPNV RIGSGSFG S151,T401,S419,S750,T753 C–Raf: RQRSTSTPNV RIGSGSFG S29,S289,S296,S301,S642

Figure 2. Regulation of the Raf kinases by phosphorylation. (Top) Raf is both positively and negatively regulated by phosphorylation on numerous sites. (Bottom) Shown are the amino acid sequence alignments of the sites of Raf phosphorylation, with the residues phosphorylated shown in red, and the relevant kinases indicated above the sequences. A glutamic acid residue that can act as a phosphomimetic to promote the active conformation of the activation segment (AS) is also shown in green.

which the amino- and carboxy-terminal do- an amino-terminal lobe (N-lobe) and a carboxy- mains of B-Raf or C-Raf were expressed as indi- terminal lobe (C-lobe) that are connected to one vidual proteins revealed that the isolated amino- another through a flexible hinge region (Shaw terminal domains of the Rafs could bind their et al. 2014). The N-lobe contains five antiparallel respective kinase domains and block signal β-strands and the regulatory αC-helix, whereas transmission to MEK and ERK (Cutler et al. the C-lobe is comprised primarily of α-helices www.perspectivesinmedicine.org 1998; Chong and Guan 2003; Tran and Frost and contains the activation segment (AS). For 2003; Tran et al. 2005). Regions in the CR1, all kinases to assume the active/ON state con- including the CRD and parts of the RBD, are formation, spatially conserved hydrophobic res- critical for the amino-terminal autoinhibitory idues spanning both lobes of the kinase domain activity, and phosphorylation of sites in the car- must align to form two structural entities called boxy-terminal kinase domain contribute prom- the regulatory and catalytic spines (McClendon inently to the relief of autoinhibition, with phos- et al. 2014). The regulatory spine (R-spine) con- phorylation of sites in the negative charge sists of four residues, with each residue derived regulatory region (N-region) being particularly from a key catalytic element in the kinase do- important (Cutler et al. 1998; Tran and Frost main—a residue from the β4-strand, the αC-he- 2003; Tran et al. 2005). lix, the DFG motif, and the HRD sequence. In In addition to the autoinhibition mediated many inactive kinase structures, the R-spine is by the amino-terminal domain, structural data misconfigured and for the spine residues to indicate that the Raf kinase domain itself is held align, both the αC-helix and the AS must change in an inactive conformation. Like all other pro- positions. Although the structure of an authen- tein kinases, the Raf kinase domain consists of tic OFF state Raf kinase has not been solved,

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E.M. Terrell and D.K. Morrison

the structure of a monomeric, inhibitor-bound held in an inactive state through intramolecular B-Raf kinase domain has been determined that interactions occurring within the kinase domain has features characteristic of other inactive pro- as well as between the kinase domain and the tein kinases (Thevakumaran et al. 2015). In this amino-terminal regulatory domain. monomeric B-Raf structure, the αC-helix is shifted to an outward position and residues ad- 14-3-3 Binding to the CR2 Site jacent to the DFG motif in the AS form a small helix called the AS-H1-helix (Fig. 3). Moreover, The autoinhibited Raf conformation is further extensive interactions between the αC-helix, the stabilized by interactions with the 14-3-3 fami- AS-H1-helix and other residues in the active ly of phosphoserine/phosphothreonine-binding site, including residues in the conserved DFG proteins (Muslin et al. 1996). All Raf kinases and HRD motifs, stabilize the αC-helix in the contain two high-affinity, phosphorylation-de- inactive OUT position and inhibit the formation pendent 14-3-3 binding sites, one in the CR2 of an essential salt bridge that defines the active (S365 of B-Raf, S259 of C-Raf, S214 of A-Raf) state (E501-K483 for B-Raf, E393-K375 for C- and one following the kinase domain (S729 of Raf, E354-K336 for A-Raf). A similarly config- B-Raf, S621 of C-Raf, S582 of A-Raf), both of ured AS-H1-helix has been observed in the in- which are phosphorylated in the presignaling active conformation of the epidermal growth inactive state (Morrison et al. 1993; Dougherty factor receptor (EGFR) kinase (Zhang et al. et al. 2004; Ritt et al. 2010). 14-3-3 proteins form 2006), and it is important to note that most constitutive dimers with each protomer of the activating B-Raf mutations identified in human dimer containing an independent binding chan- cancer occur in residues comprising the AS-H1- nel, and it has been proposed that a 14-3-3 helix (Lavoie and Therrien 2015). Thus, it is dimer can interact with both the CR2 and car- tempting to speculate that in the context of boxy-terminal sites simultaneously to stabilize full-length Raf proteins, Raf monomers are the autoinhibited conformation (Tzivion et al.

αC-helix

AS-H1 αC-helix Dimer helix interface www.perspectivesinmedicine.org

Inactive B-Raf monomer Active B-Raf dimer

Figure 3. Regulation of the Raf kinase domain by dimerization. Shown are the crystal structures of a monomeric, B-Raf kinase domain in an inactive conformation (PDB, 4WO5) and of dimerized B-Raf kinase domains in an active conformation (taken from the B-Raf–MEK complex PDB, 4MNE). In the monomeric, inactive state, the αC-helix is shifted outward and residues adjacent to the DFG motif in the activation segment (AS) form a small helix known as the AS-H1-helix. When Raf kinase domains dimerize in a side-to-side manner, the αC-helix shifts inward to the active position and the AS-H1-helix is disrupted, allowing residues in the R-spine to align. The conserved Raf dimer interface motif RKTR is shown in red, the R-spine residues in the αC-helix (L505) and in the DFG motif (D595) are indicated in yellow, and the sequences comprising the AS-H1-helix are shown in green.

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Ras-Mediated Activation of the Raf Family Kinases

1998). Moreover, binding of 14-3-3 to the CR2 was disrupted in a manner that correlated with site has a known negative regulatory function the appearance of active phosphoMEK, but and appears to mask regions within the amino could reform when phosphoMEK levels re- terminus, such as the CRD, which are required turned to baseline, suggesting that phosphory- for Raf activation (Michaud et al. 1995; Clark lation and activation of MEK disrupts the inter- et al. 1997; McPherson et al. 1999; Light et al. action. Structural analysis of the B-Raf–MEK1 2002). Mutation of the CR2 serine site or resi- complex indicates that the two kinase domains dues that comprise the 14-3-3 binding motif interact in a face-to-face manner (Haling et al. have been identified in tumors and Rasopathies 2014), with primary points of contact being the as gain-of-function mutations (Lavoie and AS of both proteins and their respective αG- Therrien 2015), resulting in increased Ras/Raf helices, a structural element implicated in ki- binding, membrane recruitment, and Raf acti- nase–substrate interactions. Although it is not vation (Michaud et al. 1995; Dhillon et al. 2002a; clear why MEK1 preferentially binds inactive Light et al. 2002; Pandit et al. 2007; Razzaque B-Raf, it is interesting to note that the RKIP et al. 2007). Protein kinase A (PKA) and AKT inhibitory protein has been found to interact have been identified as the primary cellular ki- with the C-Raf kinase domain in a manner nases that phosphorylate the CR2 site; however, that is mutually exclusive with MEK binding the CR2 site can also be phosphorylated by (Yeung et al. 2000). Phosphorylation of RKIP LATS1 through signaling cross talk with the on S153 by protein kinase C (PKC) disrupts MST/Hippo pathway (Zimmermann and Moel- the RKIP–C-Raf interaction and appears to ac- ling 1999; Dhillon et al. 2002b; Dumaz and Ma- count for the release of RKIP from C-Raf under rais 2003; Romano et al. 2014). signaling conditions (Corbit et al. 2003). From the above findings, the following model has emerged: In quiescent cells, members of Hsp90 Chaperone Complex the Raf kinase family exist as autoinhibited In their presignaling inactive state as well as in monomers, with the inactive conformation sta- their active signaling state, all members of the bilized by the phosphorylation-dependent bind- Raf kinase family interact with Hsp90 com- ing of 14-3-3 dimers as well as by the interaction plexes, which include Hsp90 proteins, the Cdc37 with Hsp90 complexes. A pool of B-Raf and targeting subunit, and the peptidyl-prolyl cis- MEK1 exists in preformed complexes, whereas trans isomerase FKBP5 (Stancato et al. 1994; MEK may be precluded from C-Raf complexes Eisenhardt et al. 2016; Diedrich et al. 2017). as a result of RKIP binding. The presignaling, The Hsp90 complex binds to the Raf kinase do- inactive Raf complexes localize to the cytosol, www.perspectivesinmedicine.org main and this interaction is required for Raf poised ready for recruitment in response to up- protein stability (Schulte et al. 1995). stream pathway activation (Fig. 4).

B-Raf–MEK1 Complex THE RAF ACTIVATION PROCESS Another protein that has been found to associate For the Rafs to become active enzymes, auto- with inactive B-Raf complexes is MEK1 (Haling inhibition mediated by the amino-terminal do- et al. 2014; Eisenhardt et al. 2016). In experi- main must be relieved and the kinase domain ments investigating whether preformed Raf/ must assume an active/ON state conformation. MEK complexes exist before Ras pathway acti- After more than 30 years of research, we now vation, MEK1 was found to preferentially inter- know that the cell accomplishes this task act with B-Raf proteins found in the cytosol of through an intricate series of events that include quiescent cells or in cancer cell lines that contain a change in Raf subcellular localization, protein low levels of active MEK (Haling et al. 2014). and lipid interactions (Fig. 1B), as well as regu- When cells were treated with growth factors to latoryphosphorylation/dephosphorylationevents activate the pathway, the B-Raf–MEK complex (Fig. 2).

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CRD CRD

Ras RBD Ras Ras GTP CR2 CR2 GTP Ras GDP RBD P P GDP

P P C

RBD B-Raf RD C-Raf Hsp90 14-3-CRD 14-3-3 RBD P

3 P Hsp90 Hsp90 MEK Fe P C-Raf phosphorylationedback B-Raf

PP1, PP2A RBD

P RKIP P Hsp90 CRD N-region kinases ERK

RBD P

P CRD P P

14-3-3 MEK C-Raf Hsp90 Signaling active state

B-Raf

MEK P Hsp90 14-3-3

Presignaling inactive state Postsignaling inactive state

Pin1, PP2A, PP5, CR2 site kinases

Figure 4. The Raf activation–inactivation cycle. A model depicting the Raf kinases in a presignaling inactive state, a signaling active state, and a postsignaling inactive state is shown (see text for details). GDP, Guanosine diphosphate; GTP, guanosine triphosphate; RBD, Ras-binding domain; CRD, cysteine-rich domain; ERK, extracellular signal-regulated kinase.

Ras Binding RBD, the CRD can further facilitate the membrane recruitment of the Rafs by selectively A major breakthrough in understanding Raf ac- binding the farnesyl groups of processed Ras tivation came with the discovery that all Raf ki- (Luo et al. 1997; Williams et al. 2000). Ras bind- nases selectively interact with active guanosine ing also allows the CRD zinc ﬁnger motifs to triphosphate (GTP)-bound Ras (Van Aelst et al. directly contact phosphatidyl serine in the plas- 1993; Vojtek et al. 1993; Zhang et al. 1993). ma membrane (Ghosh et al. 1996; Hekman et al. Binding to active Ras does not stimulate Raf 2002). Full activation of Raf requires that both enzymatic activity directly, but instead localizes the RBD and the CRD are engaged, and CRD the normally cytosolic Raf proteins to the plas- interactions with Ras and phosphatidyl serine ma membrane. This change in localization is a are critical for relief of autoinhibition as well critical step in the Raf activation process as ar-

www.perspectivesinmedicine.org as stably linking Raf to the plasma membrane tificially targeting Raf to the plasma membrane (Roy et al. 1997; Cutler et al. 1998; Bondeva et al. results in a kinase that is constitutively active in a 2002). Notably, more than 40% of activating B- Ras-independent manner (Leevers et al. 1994; Raf mutations identified in the Rasopathy car- Stokoe et al. 1994). The initial contact with dio-facio-cutaneous (CFC) syndrome occur in Ras is made by the Raf-RBD, which adopts a the CRD, further demonstrating the biological conserved ubiquitin-like structure that binds importance of this domain (Sarkozy et al. 2009). with high affinity (18 nM) to the Ras effector domain in its GTP-bound state (Herrmann et al. 1995; Nassar et al. 1995). In addition to Displacement of 14-3-3 and the RBD, the Raf CRD makes distinct contacts Dephosphorylation of the CR2 Site with lipid modified Ras. The Raf CRD is a zinc- coordinated structure of the atypical type (Mott In addition to changing the subcellular localiza- et al. 1996), and as an isolated domain, the CRD tion of the Rafs, experimental evidence suggests shows constitutive low-affinity binding to Ras that the interaction with Ras and membrane with no preference for its nucleotide state (Wil- phospholipids displaces 14-3-3 from the CR2 liams et al. 2000). However, in the context of the site, thereby exposing the site to phosphatase

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Ras-Mediated Activation of the Raf Family Kinases

activity (Rommel et al. 1996; McPherson et al. tyrosine sites are phosphorylated (Fabian et al. 1999). In response to epidermal growth factor 1993; Marais et al. 1995; Diaz et al. 1997; signaling, a complex consisting of active M-Ras, King et al. 1998; Chaudhary et al. 2000; Chi- Shoc2, and the catalytic subunit of protein phos- loeches et al. 2001; Hamilton et al. 2001; Ritt phatase 1 (PP1) plays a prominent role in me- et al. 2007). diating the dephosphorylation of the CR2 site A second major breakthrough in under- (Rodriguez-Viciana et al. 2006). However, the standing Raf activation came with the discovery CR2 site is also a substrate for the heterotrimeric that under most conditions, dimerization of the protein phosphatase PP2A (Abraham et al. Raf kinases is required. Structural studies indi- 2000; Jaumot and Hancock 2001; Ory et al. cate that the Raf kinase domains form side-to- 2003a). PP2A has been identiﬁed in active Raf side dimers with interactions mediated by both complexes and may contribute to the dephos- the N- and C-lobes of the kinase domain (Raja- phorylation of the CR2 site in speciﬁc cell types kulendran et al. 2009). Although it is unclear or under certain signaling conditions (Ory et al. whether any contacts are made between the 2003a; Eisenhardt et al. 2016). Raf amino-terminal domains, binding to Ras, concurrent with the membrane clustering of active Ras proteins, is thought to increase the local Raf Dimerization and Activating concentration of Raf at the cell surface and in- Phosphorylation duce conformational changes that promote Raf The interaction with Ras also serves a critical dimerization (Plowman et al. 2005; Terai and function in promoting the phosphorylation of Matsuda 2005; Tian et al. 2007; Hibino et al. the Raf kinase domain on activating sites and is 2009). In addition to the interaction with Ras, often a prerequisite for Raf dimerization. Phos- the retained binding of 14-3-3 to the Raf car- phorylation sites that are particularly important boxy-terminal site is also required for Raf dime- for Raf activation are found in the negative rization in cells (Rushworth et al. 2006). Wheth- charge regulatory region (N-region). The N-re- er the bound 14-3-3 dimers facilitate contact gion of all Raf proteins contains either negative- between the Raf kinase domains or act to stabi- ly charged amino acids or residues that become lize the dimerized structure is currently un- negatively charged as a result of phosphoryla- known; however, Raf proteins that contain mu- tion (Mason et al. 1999). Negative charges in this tations in the carboxy-terminal serine site fail to region contribute to the relief of autoinhibition dimerize and cannot be activated (Garnett et al. (Cutler et al. 1998; Tran and Frost 2003; Tran 2005; Rajakulendran et al. 2009; Ritt et al. 2010). et al. 2005) and are needed to form intermolec- The Raf kinases can homodimerize or heterodi- www.perspectivesinmedicine.org ular contacts that stabilize the Raf dimer struc- merize with any other Raf family member, and ture (Baljuls et al. 2011; Hu et al. 2013). The N- they can form side-to-side dimers with the regions of both C-Raf and A-Raf are comprised closely related kinase suppressor of Ras (KSR) of phosphoacceptor residues that only become proteins (Hu et al. 2013). Although the factors phosphorylated at the cell surface in response to that determine the dimerization preferences signaling stimuli (Mason et al. 1999). In con- are poorly understood, it has been shown that trast, the N-region of B-Raf maintains a constant B-Raf/C-Raf heterodimers predominate in Ras- charge because of the presence of two aspartic mediated signaling and appear to have the high- acid residues and a serine site (S445) that is con- est catalytic activity (Weber et al. 2001; Rush- stitutively phosphorylated by the CK2 kinase worth et al. 2006; Freeman et al. 2013). (Mason et al. 1999; Ritt et al. 2007). The tyrosine Structural studies have provided much in- residues in the N-region of C-Raf and A-Raf are sight regarding how dimerization promotes phosphorylated primarily by members of the Raf activation. As stated above, for protein ki- SRC family kinases, whereas the N-region serine nase domains to adopt an active/ON conforma- site can be phosphorylated by members of tion, the hydrophobic residues comprising the the PAK and PKC families, or by CK2 if the regulatory R-spine must align, and for many

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kinases, this requires that the αC-helix in the N- vitro kinase assays, one study has provided evi- lobe and the AS in the C-lobe shift to an active dence that the phosphorylation of these sites position. For the Raf kinases, the αC-helix occurs in cis after Raf dimerization (Hu et al. and the dimer interface are allosterically linked 2013). Strikingly, however, detecting these in that the hydrophobic R-spine residue in phosphorylation events in cells has been partic- the αC-helix (L505 for B-Raf, L397 for C- ularly difﬁcult. For example, these phosphory- Raf, L358 for A-Raf) lies adjacent to the con- lation sites have not been identiﬁed in any served RKTR motif in the dimer interface (Ra- large-scale phosphoproteomic study despite the jakulendran et al. 2009), and when dimerization frequent detection of other phosphorylation occurs, contacts made at the dimer interface are sites, including the Raf N-region sites (Lavoie thought to promote key conformational changes and Therrien 2015). Thus, it is possible that (Fig. 3). In particular, dimer interface contact is phosphorylation of the AS sites occurs at a thought to shift the αC-helix into the active IN very low level or is highly transient, or based position and disrupt its interactions with the on the B-Raf/MEK1 structure may not always AS-H1-helix, which in turn may help destabilize be required. In most Raf crystals the AS is largely the helix (Thevakumaran et al. 2015). Interest- unstructured; however, in the B-Raf–MEK1 ingly, a protomer does not need to possess cat- complex, residue E611 in the B-Raf AS acts as alytic activity to transactivate the second proto- a phosphomimetic to form an intramolecular mer as oncogenic mutations that abolish the salt bridge with R575 in the conserved HRD kinase activity of B-Raf can promote tumorigen- motif, allowing the AS to assume an active con- esis through the allosteric activation of C-Raf formation (Haling et al. 2014). The E611 residue (Wan et al. 2004). B-Raf proteins that are inac- aligns with AS phosphorylation sites known to tivated by inhibitor treatment can also transac- be required for the activation of other kinases, tivate C-Raf in a Ras-dependent manner to including PKCα, and given that residues analo- promote paradoxical ERK cascade signaling gous to E611 and R575 are conserved in all Raf (Hatzivassiliou et al. 2010; Heidorn et al. 2010; members, it is possible that the AS of the Raf Poulikakos et al. 2010). Notably, the key deter- kinases can adopt an “active” conformation in minant for whether a protomer in a dimer com- the absence of phosphorylation. Thus, although plex can function as an activator for the second phosphorylation of the AS sites would promote protomer is the presence of charged and/or the active conformation, their phosphorylation phosphorylated residues in the N-region (Hu may not be essential under all signaling condi- et al. 2013). tions or for all Raf proteins. Similar to many other kinases, it is also Nonetheless, it is clear that the conforma- www.perspectivesinmedicine.org thought that phosphorylation of the AS is re- tion of the AS is a critical determinant in Raf quired for full Raf activity, functioning to expose kinase activation, as many oncogenic B-Raf mu- the catalytic cleft and to promote the alignment tations occur in this segment, with V600E being of the R-spine residue in the DFG motif. Two the most prevalent. In the case of V600E, not conserved AS residues have been implicated, only would this substitution act as a phospho- T598/S601 for B-Raf, T491/S494 for C-Raf, mimetic to disrupt the AS-H1-helix, structural and T452/T455 for A-Raf, with substitution of studies indicate that the negatively charged side phosphomimetic residues at these sites resulting chain of the glutamic acid can form an intramo- in increased kinase activity (Zhang and Guan lecular salt bridge with K507, a component of 2000; Chong et al. 2001). Given that these resi- the RKTR dimer interface motif that lies at the dues localize to the region of the AS that forms tip of the αC-helix. As a result of this interaction, the AS-H1-helix, it has been proposed that both the αC-helix and the AS stably adopt an phosphorylation of these sites may be required active conformation in the absence of dimeriza- to disrupt the AS-H1-helix, which binds and tion (Haling et al. 2014; Thevakumaran et al. stabilizes the αC-helix in an inactive OUT po- 2015). Moreover, recent studies indicate that sition (Thevakumaran et al. 2015). Through in mutations to the B-Raf V600 residue are among

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Ras-Mediated Activation of the Raf Family Kinases

the few disease-associated Raf mutations that respond to a new round of activating signals. promote Raf activation in a dimerization-inde- Dephosphorylation of the ERK target sites is pendent manner (Yao et al. 2015). mediated by PP2A in a manner that requires the Pin1 prolyl-isomerase (Dougherty et al. 2004). More specifically, the WW-domain of POSTSIGNALING INACTIVATION OF RAF Pin1 binds to the pS/TP sites, allowing Pin1 to Once Raf has been activated and the signal isomerize the pS/T–proline bond to a trans con- transmitted to the downstream kinases MEK formation that can then be dephosphorylated by and ERK, it is critical that the Rafs return to PP2A. Whether PP2A also serves to dephos- an inactive state, as constitutive activation can phorylate the sites of autophosphorylation is result in tumorigenesis. Several mechanisms currently unknown; however, once dephos- that contribute to Raf inactivation have been phorylated, the Raf proteins can then reestablish identified. As expected, activating phosphory- the intramolecular contacts that mediate the lation sites must be dephosphorylated, and autoinhibited state. Finally, with the rephos- protein phosphatase 5 (PP5) has been reported phorylation of the CR2 site and the rebinding to dephosphorylate the critical S338 N-region of 14-3-3 to this site, the Raf kinases are returned site of C-Raf (von Kriegsheim et al. 2006). Raf to their presignaling, inactive conformation. autophosphorylation has also been implicated as a mechanism for inactivating the Rafs. In OTHER DIMER PARTNERS: KSR particular, a report examining inhibitor-bound B-Raf dimers has identified autoinhibitory sites Members of the KSR family are close relatives of in the ATP-binding P-loop (Holderfield et al. the Raf kinases, and like the mechanisms medi- 2013), and a phosphoproteomic study charac- ating Raf activation, the function of KSR in Ras terizing the V600E-B-Raf mutant has identi- signaling is complex. KSR was first discovered as fied an autoinhibitory site in the AS (S614- a positive modulator of the Ras pathway through B-Raf) (Dernayka et al. 2016). Further studies genetic studies performed in Drosophila and will be needed to determine the prevalence Caenorhabditis elegans (Kornfeld et al. 1995; and contribution of these autophosphoryla- Sundaram and Han 1995; Therrien et al. 1995) tion events in the context of Ras-mediated and two KSR proteins (KSR1 and KSR2) are signaling. present in mammalian cells. The KSR family Another important mechanism that attenu- shares significant sequence homology with the ates the activity of all Raf family members in- Rafs, and like the Rafs, have a carboxy-terminal volves a negative feedback loop in which active kinase domain and an amino-terminal domain www.perspectivesinmedicine.org ERK phosphorylates the Rafs on multiple S/TP that contains a CRD followed by a region rich in sites (Brummer et al. 2003; Dougherty et al. serine/threonine residues (Therrien et al. 1995). 2004; Ritt et al. 2010). Phosphorylation of the However, the KSRs do not have an RBD, but S/TP sites disrupts the interaction with Ras as instead contain a coiled-coil fused to a sterile-α well as Raf dimerization, and results in Raf pro- motif (CC-SAM) domain and a region rich in teins that are signaling incompetent (Dougherty proline residues (Therrien et al. 1995; Koveal et al. 2004; Ritt et al. 2010). Of note, some of et al. 2012). Although it was initially thought these S/TP sites are also phosphorylated by the that the KSRs would function as typical protein c-Jun amino-terminal kinases (JNKs) to prevent kinases, the mammalian KSR proteins were Raf signaling in times of cellular stress, and this found to lack a lysine residue that is normally stress-mediated desensitization of the Rafs can required for the phosphotransfer reaction (Ther- be induced by certain cancer therapeutics, such rien et al. 1995). Moreover, when residues known as rigosertib and paclitaxel (Ritt et al. 2016). to be critical for enzyme catalysis were mutated Ultimately, the postsignaling hyperphos- in C. elegans or Drosophila KSR, little to no effect phorylated Raf proteins must be recycled to a on the biological function of these KSR family presignaling inactive state that is competent to members was observed (Stewart et al. 1999; Roy

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E.M. Terrell and D.K. Morrison

et al. 2002), raising the question of whether the vation/inactivation process (Fig. 4). The early KSRs indeed possess intrinsic kinase activity—a years of research were focused on elucidating question that is still debated. the biochemical properties of the Rafs, deter- Subsequently, reports emerged indicating mining their subcellular localization, phosphor- that the KSR proteins may have scaffolding ac- ylation state, and binding partners. Subse- tivities, interacting with the three core compo- quently, the pace of discovery accelerated with nents of the ERK cascade (Morrison 2001). In the characterization of disease-associated muta- quiescent cells, KSR is found in a multiprotein tions and the analysis of Raf crystallographic complex containing MEK, 14-3-3, and compo- structures. Although much has been learned, nents of the Hsp90 complex (Xing et al. 1997; outstanding questions still remain. In particular, Denouel-Galy et al. 1998; Yu et al. 1998; Cacace it is unclear what factors determine Raf dimeri- et al. 1999; Stewart et al. 1999). Similar to the zation preferences and whether these prefer- Rafs, binding of 14-3-3 to sites in the KSR ami- ences change depending on the signaling envi- no-terminal domain sequesters the inactive KSR ronment. Moreover, the structural features of a complex in the cytosol (Müller et al. 2001; Ory full-length Raf protein are unknown as are the et al. 2003b), and as with B-Raf, the kinase do- structural changes induced by Ras binding and mains of KSR and MEK interact in a face-to-face membrane interactions. The ultimate goal is that manner (Brennan et al. 2011). On signal activa- with further insight regarding the complexities tion, KSR rapidly translocates to the plasma of Raf regulation, new strategies for therapeutic membrane coincident with the dephosphoryla- intervention will emerge for targeting Ras-me- tion of one of the 14-3-3 binding sites (Ory et al. diated Raf activation in human disease states. 2003b). The CRD and the CC-SAM domain are critical for membrane recruitment of KSR (Zhou et al. 2002; Koveal et al. 2012), and at the mem- REFERENCES brane, KSR serves to increase the local pool of Abraham D, Podar K, Pacher M, Kubicek M, Welzel N, MEK, thus facilitating MEK activation. More- Hemmings BA, Dilworth SM, Mischak H, Kolch W, Bac- over, KSR proteins can also form side-to-side carini M. 2000. Raf-1-associated protein phosphatase 2A dimers with the Rafs (Rajakulendran et al. as a positive regulator of kinase activation. J Biol Chem 275: 22300–22304. 2009; Brennan et al. 2011), and if KSR is phos- Baljuls A, Mahr R, Schwarzenau I, Muller T, Polzien L, phorylated on residues analogous to the Raf N- Hekman M, Rapp UR. 2011. Single substitution within region sites, it can function as an allosteric acti- the RKTR motif impairs kinase activity but promotes 286: – vator of Raf (Hu et al. 2013). Importantly, the dimerization of RAF kinase. J Biol Chem 16491 16503. KSR proteins also contain FxFP docking sites for Bondeva T, Balla A, Várnai P, Balla T. 2002. Structural de- www.perspectivesinmedicine.org activated ERK (Jacobs et al. 1999), and binding terminants of Ras–Raf interaction analyzed in live cells. of ERK to the membrane-localized KSR com- Mol Biol Cell 13: 2323–2333. plexes facilitates the phosphorylation of KSR Brennan DF, Dar AC, Hertz NT, Chao WC, Burlingame AL, Shokat KM, Barford D. 2011. A Raf-induced allosteric and the Rafs on S/TP sites (McKay et al. 2009). transition of KSR stimulates phosphorylation of MEK. Phosphorylation of these feedback sites disrupts Nature 472: 366–369. the signaling complexes and promotes the re- Brummer T, Naegele H, Reth M, Misawa Y. 2003. Identiﬁ- lease of KSR and the Rafs from the cell surface cation of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf. Oncogene 22: 8823– (McKay et al. 2009). Thus, KSR can modulate 8834. the dynamics of Ras pathway signaling by both Cacace AM, Michaud NR, Therrien M, Mathes K, Copeland potentiating and attenuating Raf activity and T, Rubin GM, Morrison DK. 1999. Identiﬁcation of constitutive and Ras-inducible phosphorylation sites of KSR: signal transmission to MEK and ERK. Implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression. Mol Cell Biol 19: 229–240. CONCLUDING COMMENTS Chaudhary A, King WG, Mattaliano MD, Frost JA, Diaz B, Morrison DK, Cobb MH, Marshall MS, Brugge JS. 2000. The past decades have witnessed tremendous Phosphatidylinositol 3-kinase regulates Raf1 through Pak advances in our understanding of the Raf acti- phosphorylation of serine 338. Curr Biol 10: 551–554.

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E.M. Terrell and D.K. Morrison

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14 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a033746 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Ras-Mediated Activation of the Raf Family Kinases

Elizabeth M. Terrell and Deborah K. Morrison

Cold Spring Harb Perspect Med published online January 22, 2018

Subject Collection Ras and Cancer in the 21st Century

Targeting Ras with Macromolecules MRAS: A Close but Understudied Member of the Dehua Pei, Kuangyu Chen and Hui Liao RAS Family Lucy C. Young and Pablo Rodriguez-Viciana Ras-Specific GTPase-Activating Proteins−− The Interdependent Activation of Structures, Mechanisms, and Interactions Son-of-Sevenless and Ras Klaus Scheffzek and Giridhar Shivalingaiah Pradeep Bandaru, Yasushi Kondo and John Kuriyan Ras-Mediated Activation of the Raf Family Targeting the MAPK Pathway in RAS Mutant Kinases Cancers Elizabeth M. Terrell and Deborah K. Morrison Sarah G. Hymowitz and Shiva Malek Posttranslational Modifications of RAS Proteins Ras and the Plasma Membrane: A Complicated Ian Ahearn, Mo Zhou and Mark R. Philips Relationship Yong Zhou, Priyanka Prakash, Alemayehu A. Gorfe, et al. Kras in Organoids Kras and Tumor Immunity: Friend or Foe? Derek Cheng and David Tuveson Jane Cullis, Shipra Das and Dafna Bar-Sagi KRAS: The Critical Driver and Therapeutic Target Synthetic Lethal Vulnerabilities in KRAS-Mutant for Pancreatic Cancer Cancers Andrew M. Waters and Channing J. Der Andrew J. Aguirre and William C. Hahn The K-Ras, N-Ras, and H-Ras Isoforms: Unique Efforts to Develop KRAS Inhibitors Conformational Preferences and Implications for Matthew Holderfield Targeting Oncogenic Mutants Jillian A. Parker and Carla Mattos PI3K: A Crucial Piece in the RAS Signaling Puzzle Genetically Engineered Mouse Models of Agata Adelajda Krygowska and Esther Castellano K-Ras-Driven Lung and Pancreatic Tumors: Validation of Therapeutic Targets Matthias Drosten, Carmen Guerra and Mariano Barbacid

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